Pol-? catalyzes replication of the genome and other DNA synthetic processes in human cells. However, the precise functions of Pol-? in replication, in repair of endogenous and environmental damage, and in recombination remain to be defined. Further, we lack information on the role of somatic mutations in Pol-? in tumorigenesis and other disease processes. Our goals are to identify the functions of Pol-? in DNA synthesis and the possible role of increased mutation by Pol-? in the generation of human cancer. A major approach will be to exploit mutants of Pol-? that incorporate mutagenic nucleotide analogs and thereby serve as tools to identify DNA synthesized by Pol-? in vivo. We have four specific aims.
In Aim 1, we will generate mutants of Pol-? that increase incorporation of a specific nucleotide analog(s).
In Aim 2, we will purify wild-type and mutant Pol-? holoenzyme complexes and characterize their catalytic properties in detail, including their fidelity and kinetics of analog incorporation.
In Aim 3, we will define the roles of Pol-? in mammalian cells by introducing mutant Pol-?'s that preferentially incorporate mutagenic nucleotide analogs and measuring induced mutation in cells undergoing DNA replication, repair and recombination. The induced mutations will identify the DNA synthesized by Pol-?.
In Aim 4, we will assess the role of increased mutagenesis in tumor progression by performing serial transfer experiments to determine if mammalian cells that harbor mutator Pol-? have a competitive advantage, and if there are nucleoside analogs that diminish this advantage. Narrative Our objective is to establish the roles of DNA polymerase-? in replication of the human genome and in repair of damage caused by endogenous and environmental agents. We will determine if mutations in DNA polymerase-? promote genetic instability and accelerate tumor progression in model systems.
We aim to identify a new class of chemotherapeutic agents that will retard tumor growth.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA102029-10
Application #
8239922
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Okano, Paul
Project Start
2003-07-01
Project End
2013-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
10
Fiscal Year
2012
Total Cost
$328,816
Indirect Cost
$118,036
Name
University of Washington
Department
Pathology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Shen, Jiang-Cheng; Fox, Edward J; Ahn, Eun Hyun et al. (2014) A rapid assay for measuring nucleotide excision repair by oligonucleotide retrieval. Sci Rep 4:4894
Kennedy, Scott R; Schmitt, Michael W; Fox, Edward J et al. (2014) Detecting ultralow-frequency mutations by Duplex Sequencing. Nat Protoc 9:2586-606
Fox, Edward J; Loeb, Lawrence A (2014) Cancer: One cell at a time. Nature 512:143-4
Prindle, Marc J; Schmitt, Michael W; Parmeggiani, Fabio et al. (2013) A substitution in the fingers domain of DNA polymerase ýý reduces fidelity by altering nucleotide discrimination in the catalytic site. J Biol Chem 288:5572-80
Kennedy, Scott R; Salk, Jesse J; Schmitt, Michael W et al. (2013) Ultra-sensitive sequencing reveals an age-related increase in somatic mitochondrial mutations that are inconsistent with oxidative damage. PLoS Genet 9:e1003794
Weedon, Michael N; Ellard, Sian; Prindle, Marc J et al. (2013) An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy. Nat Genet 45:947-50
Fox, Edward J; Prindle, Marc J; Loeb, Lawrence A (2013) Do mutator mutations fuel tumorigenesis? Cancer Metastasis Rev 32:353-61
Loeb, Lawrence A (2011) Human cancers express mutator phenotypes: origin, consequences and targeting. Nat Rev Cancer 11:450-7
Allen, Jennifer M; Simcha, David M; Ericson, Nolan G et al. (2011) Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication. Nucleic Acids Res 39:7020-33
Kennedy, Scott R; Chen, Cheng-Yao; Schmitt, Michael W et al. (2011) The biochemistry and fidelity of synthesis by the apicoplast genome replication DNA polymerase Pfprex from the malaria parasite Plasmodium falciparum. J Mol Biol 410:27-38

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